Mono-ADP-ribosylation, a posttranslational modification of proteins in which the ADP-ribose moiety of NAD is transferred to proteins, is responsible for the toxicity of some bacterial toxins (e.g., cholera toxin, pertussis toxin). NAD:arginine ADP-ribosyltransferases cloned from human and rabbit skeletal muscle and from mouse lymphoma cells (Yac- 1 ADP-ribosyltransferase), are glycosylphosphatidylinositol (GPI)- anchored and have similar enzymatic and physical properties. A second ADP-ribosyltransferase (Yac-2) was cloned from mouse lymphoma cells, which differs in properties from the previously identified eukaryotic transferases. The nucleotide and deduced amino acid sequences of the Yac-1 and Yac-2 transferases are 58 and 33% identical, respectively. The Yac-2 protein is membrane-bound but, unlike the Yac-1 enzyme, appears not to be GPI-anchored. The Yac-1 and Yac-2 enzymes, expressed as glutathione-S-transferase fusion proteins in E. coli, were used to compare their ADP-ribosyltransferase and NAD glycohydrolase activities. Using agmatine as the ADP-ribose acceptor, the Yac-1 enzyme was predominantly an ADP-ribosyltransferase, whereas the transferase and NAD glycohydrolase activities of the recombinant Yac-2 protein were similar. The deduced amino acid sequence of the Yac-2 transferase contained consensus regions common to several bacterial toxin and mammalian transferases and NAD glycohydrolases, consistent with the hypothesis that there is a common mechanism of NAD binding and catalysis among ADP- ribosyltransferases.